Influenza, called “flu” for short, is a respiratory infectious disease caused by influenza virus, and is characterized in clinic by high fever, feeling tired, and muscle pains, accompanied by some respiratory symptoms. Influenza virus is a threat to human health, and the sustained and rapid antigenic drift cause wide spread of seasonal influenza. Common seasonal influenza viruses in human include seasonal H1N1, seasonal H3N2 and influenza B virus. According to WHO statistics, seasonal influenza is responsible for the death of at least 250,000-500,000 persons per year (Peter D. C. et al., J Clin Invest. 2008, 118:3273-3275). In addition, flu outbreak is still an important threat to human beings. Since influenza virus was discovered, flu outbreak occurred five times around the world in human history, resulting in tens of millions of deaths; among them, Spanish influenza pandemics in 1918 resulted in about 20-50 millions of deaths in the world. The influenza pandemics occurred in the 20th century also include Asian influenza (H2N2) outbreak in 1957 and Hong Kong Influenza (H3N2) outbreak in 1968, both of which caused serious public health threat and human social panic (Xu R. et al., Science. 2010, 328: 357-360). In the 21st century, influenza outbreaks still do not stop. Influenza A (2009 pandemic H1N1) outbreak occurred in Mexico in 2009 and quickly spread around the world, which sounded the alarm to human society once again. According to WHO statistics, up to Aug. 6, 2010, a total of 18,449 cases of confirmed deaths were reported in more than 200 countries and regions globally (WHO Pandemic (H1N1) 2009—update 112. 6 Aug. 2010). When the influenza virus outbreak is over, the influenza virus will often evolve into seasonal flu and continue to prevail, and retain harmful to human health by antigenic drift in the epidemic process. In addition, human beings are also threatened by highly pathogenic avian influenza, and highly pathogenic H5N1 avian influenza virus has become one of the greatest infectious diseases that threatens human beings, after “SARS” in 2003. Since 2003, 600 cases of human infected by avian influenza virus H5N1 have been reported globally, among which 353 cases died, with a mortality rate close to 60% (WHO: http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html). Due to such a high mortality rate, people cannot help worrying that once the virus spreads in human, it will bring a fatal blow to human society. In a word, influenza caused by influenza virus is a serious infectious disease for human.
Influenza virus is an enveloped, single stranded, negative-sense RNA virus that belongs to the genus influenza virus of the family Orthomyxoviridae. Its genome contains eight segments of RNA, and encodes more than 10 viral proteins. According to the difference in antigenicity and genetic characteristics of nucleoprotein (NP) and matrix protein (M), influenza virus can be classified into Influenza A virus, Influenza B virus, and Influenza C virus (Horimoto T. et al., Nat Rev Microbiol, 2005, 3(8): 591-600). Among them, Influenza A Virus (called Flu A for short) has a broad host range, mutates fast, and can cause worldwide pandemics; Influenza B Virus (called Flu B for short) can only infect human and seals, mutates more slowly relative to Influenza A Virus, and can only cause small pandemics in local areas; Influenza C Virus (called Flu C for short) mutates the most slowly, has a weak pathogenicity, and can infect only pregnant women and children having a low resistance generally.
Influenza B virus was first isolated in 1940. Since the 1980s, for influenza B virus, the epidemic is dominated by two lineages that are quite different from each other in terms of antigenicity and genotype, i.e., Victoria lineage and Yamagata lineage. In the 1980s, the epidemic was dominated by the Victoria lineage, and in the 1990s, the epidemic was dominated by the Yamagata lineage. After the year 2000, the epidemics were dominated by both of the two lineages (Jumat M R et al., BMC Res Notes, 2014, 7: 863).
Although the flu epidemic is mainly caused by influenza A virus, influenza B virus is also an important cause for the outbreak of influenza. Among every three influenza epidemic seasons, one is dominated by influenza B virus; and influenza B virus causes diseases and death in a large number of infected patients every year (Lin et al., Virus Res, 2004, 103 (1-2): 47-52). The mortality and morbidity caused by influenza B virus were lower than those of influenza A virus H3N2 subtype, but are higher than those of H1N1 subtype (Dreyfus C. et al., Science, 2012, 337 (6100): 1343-8). Studies have shown that: the clinical symptoms caused by influenza B virus are generally not different from those of influenza A virus (Jumat M R et al., BMC Res Notes, 2014, 7: 863), and the percentage of severe cases caused by influenza B virus was not quite different from that of influenza A virus, either (Su S. et al., Clin Infect Dis, 2014, 59 (2): 252-5). In brief, in recent years, more and more studies have shown that researches on prevention and treatment of influenza B virus are of important significance in clinic.
The first line of defense to prevent influenza is neutralizing antibodies. The vaccine-induced neutralizing antibodies mainly target the protein hemagglutinin (HA) on the surface of virus. HA protein on the surface of virus has a trimeric structure, and each HA monomer consists of HA1 domain and HA2 domain. HA1 forms a globule at the top of the trimer, contains receptor binding sites, and is a region essential for the virus to infect a host cell. HA1 also contains important antigenic sites, can induce generation of protective neutralizing antibodies in organisms, and thus is a key target for vaccine design (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234). HA2 is located at the base of the trimer, has a shape of stem, contains fusion peptide, and can mediate the membrane fusion of viral envelope to a host cell. Some monoclonal antibodies against HA2 can inhibit the membrane fusion of influenza virus and achieve the virus-neutralizing effect (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234).
Influenza viruses have a high variability, particularly, HA mutates fastest. In current, traditional influenza vaccines are mainly directed to HA protein, and the influenza vaccines become ineffective easily because of virus antigenic drift caused by HA gene mutation. In order to keep influenza vaccines effective, every year WHO needs to monitor the mutation of the prevalent influenza virus strains last year, and decide to continue to use the old influenza virus vaccine strains or establish new influenza virus vaccine strains as candidate strains for influenza vaccines next year, so as to retain the effective resistance to prevalent influenza virus strains by inoculation of new vaccines every year. Therefore, development of “universal vaccines” that are not affected by virus mutation becomes a research focus for development of new vaccines. The glycoprotein on the surface of influenza virus, i.e. “hemagglutinin (HA)”, is a main target for the development of universal influenza vaccines and immunotherapeutic medicines against influenza virus. The so-called “universal vaccines” should comprise “highly conserved neutralizing epitopes” shared by different virus variant strains, and can directly induce generation of “broad-spectrum neutralizing antibodies” to combat the infection by different virus variant strains. Therefore, an important route for studying universal influenza vaccines and immunotherapeutic medicines is to look for highly conserved neutralizing epitopes on HA.
In addition, it has been demonstrated that in a mouse model, a humanized anti-HA monoclonal antibody specific for influenza B virus was able to effectively treat experimental mice infected by influenza virus (Yasugi M. et al., PLoS Pathog, 2013, 9(2)). In clinic, polyclonal antibodies and monoclonal antibodies are effective in the prevention of infections by viruses such as Hepatitis A Virus, Hepatitis B Virus, rabies virus and respiratory syncytial virus (Sawyer L. A. et al., Antiviral Res. 2000, 47: 57-77). During Spanish influenza in 1918, it was reported that serum from human in convalescent stage was used in the treatment (Luke T. C. et al., Ann Intern Med. 2006, 145:599-609). The information suggests that antibodies can be used as alternative methods and tools for anti-viral therapy.
Broad-spectrum monoclonal antibodies and highly conserved epitopes against HA of influenza A virus have been reported in many papers. As early as 2009, Throsby et al. reported for the first time that a broad-spectrum neutralizing humanized monoclonal antibody CR6261 recognizing HA2 could neutralize all the influenza viruses belonging to Group 1 (including H1, H2, H5, H6, H8 and H9 subtypes) (Throsby M. et al., PLoS One. 2009, 3: e3942). In 2011, Corti D. et al. also obtained a humanized broad-spectrum neutralizing monoclonal antibody against HA2 by using similar technology, which could neutralize 16 H subtypes of influenza viruses (Corti D. et al., Science. 2011, 333: 850-856). Yoshida et al. reported in 2008 that a broad-spectrum neutralizing monoclonal antibody S139/1 against HA1 could neutralize some influenza virus strains of H1, H2, H3 and H13 subtypes (Yoshida R. et al., PLoS Pathog. 2009, 5: e1000350).
As compared to influenza A virus, broad-spectrum monoclonal antibodies and highly conserved epitopes associated with influenza B virus were rarely reported in the literatures. Until 2012, Dreyfus C et al., found three monoclonal antibodies against HA that could neutralize two lineages of influenza B viruses, i.e., CR8033, CR8071 and CR9114 (Dreyfus C. et al., Science, 2012, 337 (6100): 1343-8). In 2013, Yasugi M et al. found another broad-spectrum neutralizing monoclonal antibody 5A7 against different lineages of Influenza B viruses (Yasugi M. et al., PLoS Pathog, 2013, 9 (2)).
Most of the epitopes recognized by the monoclonal antibodies obtained in the above researches were located in a conserved region near the fusion peptide on HA2 (the primary function of which was to mediate the membrane fusion of influenza virus), instead of HA1 domain having immunodominance in recognition. It increases the uncertainty in the application of these antibodies and the conserved epitopes recognized thereby in the prevention and treatment. Some studies show that the neutralizing monoclonal antibodies recognizing HA2 have the activity of neutralizing natural viruses reduced by 100-1000 folds relative to the activity of neutralizing the pseudotype viruses, the reason may be that it is not easy to have the HA2 epitope of natural virus exposed, and therefore the epitope is not easily accessible (Sui J. et al., Nat Struct Mol Biol. 2009, 16: 265-273; Corti D. et al., J Clin Invest. 2010, 120: 1663-1673).
As compared to HA2, HA1 of influenza virus forms a globule at the top of the trimer, contains a lot of neutralizing epitopes, and is easily accessible. Therefore, looking for broad-spectrum neutralizing epitopes on HA1 is beneficial for development of highly effective broad-spectrum influenza vaccines and therapeutic antibodies. Cyrille Dreyfus et al. reported in 2012 the only one broad-spectrum monoclonal antibody CR8033 so far that recognizes HA1 domain of influenza B virus, however, the monoclonal antibody had hemagglutination-inhibiting activity only against the Yamagata lineage of influenza B virus (Dreyfus C. et al., Science, 2012, 337 (6100): 1343-8.). Therefore, it is of importance and instructive significance for development of broad-spectrum therapeutic antibodies and universal vaccines against influenza B virus, to develop broad-spectrum neutralizing monoclonal antibodies that recognize more highly conserved neutralizing epitopes on HA1 of influenza B virus, and to accurately localize the epitopes. There is need in the art to develop more broad-spectrum monoclonal antibodies that recognize more highly conserved epitopes of HA1 of influenza B virus.